Inhibition of HPV-16 E6/E7 immortalization of normal keratinocytes by hairpin ribozymes

Abstract

HPV-16 E6 and E7 genes are required to efficiently immortalize a broad spectrum of cell types including cervical keratinocytes. Therefore, the E6͞E7 genes can be considered relevant targets for anti-cancer therapy. We produced several engineered hairpin (HP) ribozymes to specifically disrupt HPV-16 E6͞E7 mRNA. After extensive biochemical characterization, one anti-E6 HP ribozyme (R434) was selected for in vivo testing because of its superior catalytic capabilities. When expressed in cis, R434 efficiently inhibited E6 in vitro translation. Cis-expression of the HP ribozyme with HPV-16 E6͞E7 genes in normal human keratinocytes reduced the growth rate and prevented immortalization. RNA analysis by reverse transcription-PCR showed that E6͞E7 transcripts were cleaved in post-transfected cells and virtually were eliminated after long term expression. Of interest, an inactive version of the HP also was able to significantly affect the immortalizing ability of E6͞E7, probably through passive hybridization. The combination of passive and cleaving antisense RNA therefore is established as an effective inhibitor of HPV-16 E6͞E7 immortalization.Over one-half of invasive cervical carcinomas worldwide and many cervical carcinoma-derived cell lines contain and express DNA from human papillomavirus type 16 (HPV-16). In early stages, HPV-16 expression causes benign proliferation and efficiently immortalizes cultured human epithelial cells, including cervical keratinocytes (1-3). The HPV-16 viral, early genes E6 and E7 are required to acquire and maintain efficiently the transformed phenotype in a broad spectrum of cell types and commonly are expressed in cervical carcinoma cells. Thus, E6͞E7 genes are the hallmark of cervical carcinoma (4-7). The E6 and E7 proteins bind with high affinity the p53 and Rb tumor suppressors, respectively (8, 9). The interaction of HPV-16 E6 protein with p53 results in degradation through the ubiquitin pathway, resulting in the equivalent of a mutant p53 phenotype (10-12). The association of E7 with Rb impedes the interaction of Rb with several proteins (i.e., E2F), efficiently disrupting the cell cycle (13-15). Therefore, the E6͞E7 genes are ideal targets for anti-cancer therapy.Antisense RNA and oligonucleotides have been used specifically to block translation of several genes. This effect is obtained by the hybridization of passive antisense molecules with their respective complementary mRNA to form nontranslatable double-stranded RNA molecules or DNA-RNA hybrids that promote the activity of endogenous RNase H, an enzyme that specifically digests the RNA strand of DNA-RNA hybrid molecules (16)(17)(18). In cultured tumor cells, antisense oligonucleotides have been shown to suppress effectively translation of several genes and to reverse some phenotypes (19)(20)(21)(22). However, passive antisense therapy has the disadvantage of being active for a limited period and often causing nonspecific toxicity (23,24). This approach has produced inhibition of E6͞E7 gene expression in HPV-18-containin...

Induction of apoptosis in human papillomaviruspositive cancer cells by peptide aptamers targeting the viral E6 oncoprotein

“…Induction of apoptosis, rather than growth inhibition, would be particularly desirable for therapeutic agents, because such agents may not require continuous application. It is noteworthy that in previous attempts to inhibit HPV oncogene expression, e.g., by antisense constructs or ribozymes directed against the polycistronic E6͞E7 mRNA, (partial) growth inhibition of HPV-positive cells was reported, rather than induction of apoptosis (41)(42)(43)(44)(45)(46). This result could be due to the concomitant reduction of proapoptotic E7 levels, which has been observed even for antisense constructs directed selectively against the E6 portion of the polycistronic E6͞E7 transcripts (43,46).…”

HPV E6 specifically targets different cellular pools of its PDZ domain-containing tumour suppressor substrates for proteasome-mediated degradation

“…Perhaps the most important of these are those associated with the development of cervical cancer, of which HPV-16 and HPV-18 are the most common (zur Hausen, 1996). Both viruses encode two major transforming proteins, E6 and E7 Mu¨nger et al, 2001 for reviews), the continued expression of which is required for the continued proliferation of tumour-derived cell lines (von Knebel Doeberitz et al, 1992;Alvarez-Salas et al, 1998;Butz et al, 2000). A great deal of emphasis has been placed on defining the mechanism of action of these two viral proteins and, in particular, in identifying the cellular target proteins with which they interact.…”

Highly specific targeting of HPV genes using ribozymes

“…These include strategic design to optimize specificity and turnover, suitable delivery mechanisms and the enhancement of stability. The use of ribozymes for the effective cleavage of HPV transcripts 14 and in the inhibition of E6/E7-mediated immortalization 15 has been shown previously. HeLa cells carrying ribozymes against HPV 18 have been shown to exhibit reduced growth rates, increased serum dependency, and reduced focus formation in soft agar.…”

“…The introduction of tumor-suppressive or pro-apoptotic genes such as p53 3,4 and its homologues, 5 Rb, 6 bax 7 and MCP1, 8 cell cycle inhibitors such as p21 WAF/CIP1 , 9 and more recently, antisense RNA, 10-12 siRNA 13 and ribozymes [14][15][16] against viral genes, have been found to be effective in inhibiting the growth of cervical cancer cells.…”